Bacteria have been used to produce a wide range of commercial products. The use of genetically engineered bacteria like E. coli as host cells for the production of biological reagents such as peptides and nucleic acids in the laboratory and industry, is well-known in the art. Bacteria in their natural environment are exposed to many conditions that are not normally experienced in standard industrial or laboratory growth, and thus their genomes carry a large number of condition-dependent, stress-induced genes or otherwise non-essential genes which may not be needed in industrial or laboratory use of the organisms, or may even be unwanted.
Bacterial pathogens contain virulence factors such as capsular polysaccharides at the cell surface, which mediate the interactions between the bacterium and its immediate environment and have a number of functions directly related to their pathogenicity. More than 80 serologically and chemically distinct types of polysaccharide capsules have been described in E. coli and termed K antigens. The polysaccharide capsules have been classified into 4 groups, of which the group II capsular polysaccharides are similar to those of Haemophilus influenzae and Neisseria meningitides. The group II capsular gene clusters contain most of the capsular types associated with invasive disease and have a conserved genetic organisation consisting of three functional regions. Regions 1 and 3 are conserved among group II gene clusters and encode peptides necessary for the transport of the polysaccharide from its site of synthesis to the cell surface, while region 2 is serotype specific and encodes the enzymes responsible for biosynthesis and polymerization of the individual monosaccharides that comprise the particular polysaccharide. Region 1 comprises 6 genes kpsFEDUCS, organised in a single transcriptional unit. Region 2 is serotype specific and differs among group 2 antigens. Region 3 contains 2 genes kpsM and kpsT organised in a single transcriptional unit [Andreishcheva et al. Gene (2006) 384: 113-119].
The E. coli strains K-12 and B are not encapsulated and are commonly used strains for the production of peptides. The E. coli K-12 genome has been fully sequenced and does not contain any capsular genes. It has been found that E. coli B BL21(DE3), which is a preferred strain for high-yield expression of recombinant peptides, contains a cluster of genes coding for group II capsular polysaccharides and it is thought that an ancestor of E. coli B must have acquired these capsular genes by horizontal transfer. The K antigens located in the capsule of E. coli are often associated with a pathogenic phenotype in strains harbouring the functional form of the genes. It has been shown that in E. coli B BL21, the region 2 of group II capsular gene cluster has become inactive by an insertion element (IS1), preventing the production of the antigen, while region 1 and 3 supporting export and presentation of wildtype capsular antigen, are still intact [Andreishcheva et al. Gene (2006) 384: 113-119]. However, there is a theoretical possibility of re-establishing a functional group II capsular gene cluster, e.g. if the IS1 in region 2 is lost and thereby the group II cluster may be re-established. Alternatively, if the IS1 mutated region is exchanged by an exogenous wildtype region 2, the group II cluster may also be re-established.
The present invention provides an E. coli B BL21 (DE3) strain with permanent knock out (KO) of the group II capsular gene cluster, bringing this strain into the same safety class as other commercially important strains such as E. coli K-12.